Proteinase inhibitors are molecules acting in normal control mechanisms of the proteolytic enzyme activity and are related to many physiological processes as for example coagulation, fibrinolysis, digestion and also in pathologies as cancer, hemorrhagic disturbances, inflammation and blood pressure balance (DECLERCK & IMREM, 1994).
One of the most efficient means for controlling coagulation is the action of proteinase inhibitors. Studies have demonstrated that salivary glands of the majority of the hematophages species as ticks for instance, produce several anti-coagulant substances aiming to turn blood more fluid then optimizing its feeding. (RIBEIRO, 1995).
The inhibitors of Factor X activated (FXa) are of great clinical interest, since through FXa inhibition (in the prothrombinase complex) the control of the activation of the prothrombin into thrombin is possible avoiding clot formation.
The inhibition of proteases is trigged as soon as starts the coagulation process. The “Tissue Factor Pathway Inhibitor” (TFPI) is the main inhibitor of the extrinsic path and was classified as a member of the Kunitz family inhibitors of BPTI (Bovine Pancreatic Trypsin Inhibitor) type (Broze, 1998).
The human TFPI is a protein composed of three Kunitz-type domains (K1, K2 and K3), and K1, the first domain, has an acid region at the N-terminus portion where the binding site to the FVIIa is placed. In the second domain we can find the area responsible for the FXa binding and finally the third domain presents a basic region at its C-terminus portion of which function has not yet been elucidated but probably contains a heparin binding site (Rao, 1995).
The human TFPI inhibition mechanism comprehends two phases. In the first phase the inhibitor associates itself to factors FXa and FVIIa, however the inhibition will really occur in the following phase where a formation of a quaternary complex is seen with the presence of Tissue Factor (TF), FXa, FVIIa and human TFPI (TFPI: FXa: FVIIa: TF) (Sandset & Bendz, 1997).
The inhibitor of the human tissue factor type-2 “tissue factor pathway inhibitor-2” (TFPI-2), is a protein of 32-kDa consisting in three Kunitz-type domains (Chand et. al., 2004). TFPI-2 inhibits a variety of serine proteases involved in coagulation and fibrinolysis probably due to the arginine residue found in the position 24 (R24) of the first Kunitz-type domain. In the second and third domains residues of glutamin and serine respectively can be found. In recent studies a mutant was built where the arginine residue was modified by a glutamin residue (R24Q TFPI-2) and this mutation originates around 90% of the inhibiting activity loss on bovine trypsin. This fact demonstrates the importance of this residue in maintaining the inhibitory activity (Kamei et. al., 1999).
General Aspects of Cancer Development and its Interactions with Coagulation
The risk of a subject developing a neoplasia is determined by a combination of several genetic and environmental factors. Therefore, in the carcinogenesis process, the substance called carcinogen (of biological, chemical or physical natures), can act as starter or even promoter of the process that develops up to the formation of metastases (Ruoslahti E., 1996).
In the starting of the neoplasia process irreversible alterations occur in target structures of the DNA, contributing for the cellular transformation. The carcinogen metabolism is shown in two phases called Phase I (activation) and Phase II (detoxification). Some Phase I enzymes act not only as catalyzer of oxidative reactions, but also metabolizing high quantities of carcinogens or lipophilic xenobiotics (Bell et. al, 1993).
The persistency of the DNA damages depends on repairing mechanisms and on the cellular life lasting of the damaged tissue, that way, if the damage persists or is not repaired, there is an expansion of a mutant cellular clone (Duke et al, 1996; Wainscoat e Fey, 1990).
It is a process, long in many times that occurs on started cells, decreased in latency period and/or increasing their susceptibility to the genetic alterations. Usually the promoters are not genotoxic. They are specific-tissue and have multiple action mechanisms acting in an epigenetic form resulting in tissue homeostasis disturbances (Hermo et al, 1987).
The promotion mechanisms include the activation of cellular surface receptors, activation or inhibition of cytosolic enzymes, activation of transcription and translation factors (by Kinase), proliferation stimulation, apoptosis inhibition, and direct cytotoxicity.
In the progression stage, the tumoral cells also show capacity of forming new blood vessels which will feed them, provoking uncontrolled increasing since they invade tissues around them at first and can be reaching the inside of a lymphatic or blood vessel and through them be spread into other organs. (Meyer et. al., 1998, Matsuda et al 2003).
The cells usually react to several intrinsic damages generated from intermediate metabolism products, from severe or chronic inflammatory reactions and from process causing oxygen and nitrogen unstable reactive metabolites. Besides that, there are extrinsic-damaging factors as for example physical, chemical and biological agents eliminated by homeostatic process.
The last stage, (called tumoral progression), includes invasion and metastasization. In this phase, pre-existing or pre-neoplasia lesions are added to aleatory mutational alterations including aberration of specific-sequence, duplication, deletion and/or loss of heterozygosity in specific genes as oncogenes, tumor suppressor genes, metastogeneses and repairing genes.
Oncogenes are inactive in physiological conditions and can be activate by changing an amino acid, or by the amplification of a gene in a chromosome originating several copies of this gene with the increase of its activity and finally, by the recombination among genes of distinct chromosomes. The difference of these genes is that they are usually active, vigilant for avoiding the uncontrolled growing of the cells (Budillon, 1995).
Although the genomic instability is the main characteristic of the tumoral progression, it is the absence of the control of cellular duplication what distinguishes the malignancy levels and turns them into lethal cancers. The tumoral cells as well as those of a normal tissue duplicate through the cellular cycle (Fearon E R, 1997).
To understand cellular cycle, however, have not completely cleared up the regulation mechanism of the cellular multiplication of tumoral cells and cancers growing characteristics in vivo have not yet been elucidated.
The invasive nature of the tumoral progression is associated to the increase of mobility of tumoral cells, to the proteolysis capacity and to the loss of inhibition of cell-cell and cell-matrix contact. The metastatic cells are then disseminated through the circulatory, lymphatic systems by local extension or even by the implanting process. Therefore, standards for metastasis locations will vary depending on the kind of primary cancer and on organs since some of them like muscles, skin, thymus and spleen will rarely present metastases (Goel, et al., 2004).
Some cancers considered untreatable or without therapeutical response predominantly repeat the place of the primary cancer and in its region lymph nodules. These tumors can also produce metastases during the treatment.
The incidence of malign tumors represents a significant number ranking as the second cause of death in the world. Cancer treatment is based, in general, on surgical removing of solid tumors placed in situ, radiotherapy for tumors in patients without clinical conditions or technical possibilities for its complete removing and chemotherapy for cases of non-solid tumors or of solid tumor spreading.
Chemotherapy, radiotherapy and surgery turned to be therapeutical methods of intensive administration associated with other treatments and introducing the concept of adjuvant treatments has been an innovation in the process of control and cure of many sick patients, improving their results (Tsao, et. al., 2004).
Today, more accurate radiotherapeutical protocols involve the administration of higher doses in tumoral mass, and less intense in healthy adjacent tissues; chemotherapy treatments with efficient drugs on the neoplasia and less side effects allowing wider oncology conducting and consequently more encouraging results for many cases. However, the majority of solid tumors presents modest or inefficient responses to chemotherapy, limiting indications and efficiency both in adjuvant treatment on local tumors and in therapeutical of metastasis cases (Sekire, et al., 2004).
The high incidence of metastases occurring in 50% of patients with advanced lung cancer shows how necessary it is to develop new and more efficient therapeutical strategies aiming to offer to these patients' real opportunities for controlling the proliferation and dissemination of the neoplasia cells.
In the twentieth century we reached an extraordinary advance toward it when new drugs were invented containing wide anti-neoplasia proprieties as the nitrogenated mustard identified in the 40's and a variety of other substances with partial or complete efficacy against some histologic kinds of tumors. Although there is a fast expansion of the anti-cancer drug selection allowing treating several solid tumors like lung, colon, breast and prostate cancers, there has not yet been a systemic treatment proving to be adequate and efficient toward that. In lung carcinomas of non-small cells and in skin melanomas, prevalent in developed countries however frequent all over the world, the modest response when experiencing chemotherapy is from 15% to 20% in all available protocols and from 40% to 50% in other therapeutical associations (Sekire, et al., 2004).
Therefore, searching for new drug alternatives for improving treatment efficacy on advanced neoplasia diseases is vital. The majority of the chemotherapies are known for their capacity of controlling cellular proliferation and drugs with specific activity against some metabolic mechanisms exclusive for tumoral cells (target treatment) were recently identified. In these treatments the substances act against the tumors through cellular cycle alterations allowing being more efficient right in their action mechanisms.
Cancer morbidity related to chemotherapy treatments is still a significant obstacle, therefore, finding anti-neoplasia drugs of easy administration with few or insignificant side effects and selective action mechanisms for the neoplasia cell seems to be a target today.
One of the regulatory routes involved in tumoral growing and progression is the blood coagulation system where primarily this response is caused by the expression of pro-coagulating factors as the Tissue Factor (TF), one of the proteins that trigger the coagulation process leading to thrombin production and clot formation.
At a second phase, tumoral cells come to express the receptor of the urokinase-type plasminogen activator (u-PA) and the plasminogen activators promote the activation of plasminogen into plasmine. This system is responsible for the tissue coordination and remodeling.
In patients with neoplasias as for example pulmonary adenocarcinomas, renal carcinomas and in malign melanoma, there is a loss of homeostasis provoking alterations of the coagulation system, arising from thrombin production and Factor X activated (Edward R L, 1993). On the other hand, the blood coagulation activation occurring in patients with primary neoplasias, or submitted to chemotherapeutic treatments is influenced by the biology of the tumoral cells of which mechanisms have not yet been understood (RicKels et. al., 1992, Schafer, et al., 2003).
The laboratory evaluation of coagulation parameters has shown high levels significant in certain kinds of tumors, turning possible determining it in circulation as for example the presence of prothrombin fragments, of D-dimers, of Factor XIIa and its prognostic correlation (Gordon S G, 1992).
The treatment of patients with primary tumors, metastases or presenting damages during the chemotherapy treatment, with anticoagulants as for example the low molecular weight heparins or K vitamin inhibitors (Hoffmal et al., 2001) showed efficiency in prolonging the life of patients (Moussa et. al., 2003; Sutherland et. al., 2003). The pathogenesis of venous thrombosis in malign neoplasias is multifactorial and its mechanism involves the releasing of pro-coagulant components by the tumor, genetic factors and even of the treatment itself (Sorensen et. al., 2003; Gouin-Thibaut e Samama M M, 2000).
Clinical studies have demonstrated that this thromboembolic feature can be prevented by the use of low molecular weight heparins in breast cancers removed by surgery (Bergqvist D., 2002; Lee e Levine, 2003). The histologic and functional evidences of the interaction of several biological systems as immune system and coagulation in certain neoplasias were identified. They revealed important alterations as the presence of coagulation activating factors associated to the inflammatory infiltrated, to the endothelial cells and the neoangiogeneses (Loreto et. al, 2000; Zacharcki D, 2003; Ornstein et. al, 2000; Gale e Gordon 2001; Cotrino J, 1996).
Cytokines derived from tumoral cells as tumoral necrosis factor (TNF), interleukin 1 and the vascular endothelial growing factor (VEGF), are chemotactic and induce vessel formations since they are able to induce the expression of pro-coagulant and thrombogenic factors, modifying the pro-coagulant state at the tumoral micro ambient (Sorensen, et. al, 2003, Fernandez et. al, 2004).